FIGURE 1.

Silencing IRS-2 inhibits the response of cultured hepatocytes to insulin and IFNα. A, in HepG2 cells, IRS-2 protein expression was silenced, and RT-PCR was performed for IRS-2 (IRS-2 cDNA) and GAPDH (GAPDH cDNA) transcripts. Decreased IRS-2 protein expression was examined by Western blot. B, control (siRNA Control) and insulin-resistant cells (siRNA IRS-2) were treated with 100 nm insulin for 15 min in the absence or presence of 0.1 mm pervanadate (PV). Whole cellular proteins were analyzed by Western blot using specific antibodies against Akt or serine-phosphorylated Akt (pAkt). Control, untreated cells. C, control and insulin-resistant cells were treated with 250 units/ml IFNα for 60 min in the absence or presence of 0.1 mm pervanadate. Cellular proteins were immunoprecipitated (IP) with anti-Stat-1 antibody, and total and tyrosine phosphorylated Stat-1 levels were analyzed by Western blot (WB). D and E, 2′5′OAS (D) and Mx (E) gene expression was measured by quantitative RT-PCR using endogenous GAPDH as reference. Control cells and cells with silenced IRS-2 were treated with IFNα in the presence or absence of 0.1 mm pervanadate. These blots are representative of experiments that were repeated three times. ***, p < 0.001; NS, not significant.